1. Field of the Invention
The invention relates to a process for preparing human immunoglobulin concentrates for therapeutic use, from plasma or a fraction of human plasma. The process enables immunoglobulins G (IgG), immunoglobulins A (IgA) and immunoglobulins M (IgM) to be obtained.
2. Related Prior Art
The use of human plasma fractions enriched with immunoglobulins for the treatment of various infections or congenital deficiencies has been known since the development of the ethanol precipitation process by Cohn (Cohn et al. 1946, J. Am. Chem. Soc. 68, 459; Oncley et al., 1949, J. Am. Chem. Soc. 71, 541). As the therapeutic indications for immunoglobulins have grown in number, there is a need for a product offering ever increasing performance and purity.
The complexity of the structure of immunoglobulins (four polypeptidic chains joined by disulphide bonds) and the variety of antibodies present in the mixture of the plasma of several thousand donors are not presently factors favouring the biotechnological development of immunoglobulins. Although some monoclonal antibodies are produced by genetic engineering, their extreme specificity represents a drawback for therapeutic applications in which polyreactivity appears necessary.
In addition, numerous pathologies, in particular of autoimmune origin, are presently treated using IgG concentrates. This widespread use has led to a shortage in Europe and the United States over the last few years.
Furthermore, in these same pathologies, the efficiency of IgM-enriched preparations has recently been demonstrated (Hurez et al. Blood 90, 1997, 4004–4013), but there exists no preparation for therapeutic use that is sufficiently purified and that has a sufficient IgM concentration.
This is why the Applicant has devoted itself to the development of a new process for the preparation of human immunoglobulins. The process can be applied to a pool of serums (from at least 10 000 donors), which ensures the presence of all the antibodies normally present throughout the population of a chosen region, or to hyperimmune serums selected for their specific immunoglobulin content. The process further allows the preparation of IgA and IgM concentrates.
Numerous variants of the original Cohn process have been described. They propose, in addition to the selective precipitation of the proteins with ethanol, various additional treatments such as precipitation with polyethylene glycol, the gentle treatment with proteolytic enzymes, etc., intended to eliminate the aggregates of immunoglobulin polymers (liable to activate the complement system and to lead to anaphylactic reactions).
An alternative approach to ethanol precipitation is described by Steinbuch et al. (Rev. Franc. Et. Clin. et Biol. 1969, XIV, 1054); this approach uses precipitation with octanoic acid. This acid precipitates most of the proteins in the plasma and leaves the immunoglobulins in the supernatant. Purification of these immunoglobulins is continued by adsorption (in “batches”) using an anion exchanger, DEAE-cellulose, which also leaves the immunoglobulins in the supernatant. The latter is then concentrated.
Various processes have also been developed for increasing the purity of the products through the use of chromatographic separation processes. Those yielding the best performance (in particular EP 0 703 922, WO 99/64462) include at least two successive chromatography steps, one using anion exchange and the other using cation exchange. The specificity of these processes is provided by the property of the anion exchangers of not adsorbing the immunoglobulins G, under conventional conditions of implementation, but of fixing most of the other proteins co-purified during the pre-purification steps.
Various purified IgG preparations are thus already available but their preparation processes still pose problems in terms of productivity and complexity of implementation on an industrial scale. These problems are further increased by the need to include in the process viral inactivation, involving an additional step to eliminate the virucidal agents used.
In addition, all of the processes presently described have been developed and optimised for the production of IgGs only.